Before and at the clinical onset, insulin-dependent diabetes mellitus (IDDM) is closely associated with islet auto-immunity, in particular with the presence of auto-antibodies (Ab) against GAD65 - the Mr 65,000 isoform of glutamic acid decarboxylase. GAD65Ab have a high diagnostic sensitivity, specificity and predictive value for IDDM. The now standardized GAD65Ab radiobinding assay that we successfully developed after cloning human islet GAD65 using our GAD65 cDNA plasmid to prepare radioactive GAD65 by coupled in vitro transcription and translation was used to demonstrate that epitope-specific GAD65Ab have a higher diagnostic sensitivity for IDDM than non-epitope analyzed GAD65Ab. The objective is to determine the mechanisms of GAD65 antibody-associated auto-immunity in IDDM in two Specific Aims. 1) To test the hypothesis that epitope-specific GAD65Ab determine progression to IDDM in intermediary phenotypes. Specifically, we will construct combinatorial antibody libraries from: 1) new onset IDDM patients (Project 3); 2) GAD65Ab- positive healthy individuals not progressing to IDDM (Project 4); 3) GAD65Ab-positive patients who are classified and treated for type 2 (NIDDM) diabetes (Project 3) in order to isolate Fab reactive with IDDM-associated GAD65 epitopes; b. determine the cDNA sequences of heavy and light chain anti- GAD65Ab selected by GAD65 epitope-specific panning. 2) To test the hypothesis that GAD65 antibody-mediated uptake of GAD65 deviate HLA- restricted clonal T cell responses. Specifically we will: a. transfect epitope-specific GAD65Ab to human B cells to determine uptake, processing and presentation; b. explore the role of antibody fine specificity in influencing GAD65 epitope presentation using GAD65 peptide and HLA restricted T cell clones in collaboration with Project 1. Project 2 will collaborate with project 1 to define the mechanisms by which membrane-bound GAD65Ab on B lymphocytes may deviate antigen- presentation on MHC Class II antigens and subsequent T-cell responses. GAD65-peptide specific and DRB1 or DQB1-restricted T cells from project 1 will be used to determine deviation responses. The collaboration with Project 3 is to study new onset IDDM patients and GAD65Ab-positive patients who are classified and treated for type 2 (NIDDM) diabetes. These patients will be compared with non-progressing marker-positive healthy subjects individuals from Project 4.